A Front-end Desaturase from Chlamydomonas reinhardtii Produces Pinolenic and Coniferonic Acids by omega 13 Desaturation in Methylotrophic Yeast and Tobacco

Pinolenic acid (PA; 18:3 Delta super(5,9,12)) and coniferonic acid (CA; 18:4 Delta super(5,9,12,15)) are Delta super(5)-unsaturated bis-methylene-interrupted fatty acids ( Delta super(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2 Delta super(9,12)) and alpha -linolenic acid (ALA; 18:3 Delta super(9,12,15)), respectively, by Delta super(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta super(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta super(5)-desaturase activity on 18:1 Delta super(9) but not 18:1 Delta super(11). Unexpectedly, CrDES also showed Delta super(7)-desaturase activity on 20:2 Delta super(11,14) and 20:3 Delta super(11,14,17) to produce 20:3 Delta super(7,11,14) and 20:4 Delta super(7,11,14,17), respectively. Since both the Delta super(5) bond in C18 and the Delta super(7) bond in C20 fatty acids are ' omega 13' double bonds, these results indicate that CrDES has omega 13 desaturase activity for omega 9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.